Overexpression of a protien (fwd)

Tomas Drgon tomasd at bdg10.niddk.nih.gov
Fri Oct 27 11:12:17 EST 1995


In article
<Pine.SOL.3.91.951025101344.12308B-100000 at welchlink.welch.jhu.edu>,
tlu at WELCHLINK.WELCH.JHU.EDU (Natalie Lu) wrote:


> I have problems to overexpress my protein. The gene (2kb) was 
> successfully cloned into pET21-a(+) vector. The cloned gene was excised 
> by the same two enzymes (Nde I and XhoI). I saw  the insert on the gel 
> having the correct weight. The DNA sequence of the cloned plasmid is 
> perfect. The insert was cloned into the NdeI site of the vector. Then,the 
> the plasmid was tramsformed into BL21(DE3) cells. The clone was picked to 
> test the overexpression of the protein. I didnot see any overexpressed 
> protien at all. The conditions for grow the cells is as follows: LB 
> medium containing 100ug/ml ampicillin, when O. D. reaches 0.4-0.6, 100mM 
> IPTG was added to a final concentration of 1 mM. Aliquots (500ul) culture 
> was removed every one hour upto 5 hours. The left culture was allowed to 
> grow overnight.No major protien band was found on the SDS gel compared to 
> the uninduced control.  If anyone has any suggestions about this, I will be 
> appreciated. Thanks.

Hi Natalie.
My experience is, that whatever you do, only about one third of constructs
which should overexpress a protein really do it. I did about 20 of them. I
think the only way is to try many different constructs. Maybe a good trick
is to use a vector, where you can fuse your protein to some other, which
is readilly expressed (like DHFR). There, if the expression of your
protein is stopped for some reason, you still see the DHFR piece, which
ensures you, that at least your expression conditions are ok. Check out
the Qiaexpress system by Quiagen. It worked well in my hands.

(do not work for Qiagen)

Tomas

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